1. Field of the Invention
The present invention relates to the engineering of heterologous gene constructs by recombinant DNA techniques for the more efficient processing and secretion of heterologous genes in eukaryotic cells. Particularly the present invention relates to functionally linking the heterologous gene in frame to the 3' end of the juvenile hormone esterase gene.
2. Description of the Related Art
Recombinant polypeptides for medical, research and veterinary applications are produced using a wide variety of genetically engineered organisms that include transgenic animals (eg. cows, goats) transgenic plants (eg. canola) recombinant viruses (eg. baculoviruses) and transformed prokaryotic cells (eg. bacteria) and eukaryotic cells (eg. yeast and animal cells) in culture.
Since most of the proteins are glycoproteins requiring advanced post-translational modification expression systems using yeast and bacteria are unsuitable. For this reason, other protein expression systems were developed using higher eukaryotes, including virus-based expression systems such as baculovirus and adenovirus and expression from transformed mammalian cells (CHO, BHK NsO etc. and production in the milk of transgenic farm animals). However, even these most advanced vehicles for protein production are inadequate due to difficulties in recovery and purification of the recombinant proteins.
Viral expression systems can produce impressive levels of recombinant proteins in both insect (Maiorella et al. 1988) and mouse cell lines (Garnier et al., 1994) but suffer from serious biological and engineering disadvantages. First, because host cells are killed at the end of each infection cycle, protein expression is only temporary. This also means that protein expression is not suited to the state of the art perfusion bioreactors. Second, the biological authenticity of the expressed protein is not guaranteed because the cell machinery necessary for post-translational modifications is inactivated in the late stages of infection. Unsuitable N-linked glycosylation patterns are widely reported for proteins produced following infection with recombinant viruses, which alters the normal glycosylation characteristics of the cell hosts (Jarvis and Summers, 1989). It is known however that the lepidopteran insect cell hosts are capable of the complex oligosaccharide processing required for in vivo human use of proteins (Davis and Wood 1995) Thirdly, although native genes containing all or part of their introns are generally expressed at a higher level than the corresponding cDNAs (Brinster et al. 1988) virus infected insect cells cannot efficiently excise introns from expressed genomic DNA, thus limiting foreign protein expression from cDNAs only (O'Reilly et al., 1991). Fourth, purification of recombinant proteins from virus infected systems is problematic. Because proteins cannot be secreted efficiently in viral systems due to the inactivation of the secretory pathway upon infection (Jarvis and Summers 1989) they must be recovered from cell lysates after cell lysis. The presence of proteases in such cellular lysates also cause degradation of the over-expressed product.
A major problem in biotechnology exists in the production and recovery of recombinant non-secretion competent polypeptides, such as intracellular proteins or protein subunits, from genetically engineered organisms. Often these intracellular proteins or protein subunits can be expressed at only moderate levels inside a cell and their purification must first include steps to lyse the cells, followed by complex procedures to isolate the desired polypeptides from many other intracellular proteins.
All secreted proteins possess a consensus signal peptide of 10 to 50 amino acids at their N-terminus that directs the protein to the secretory pathway of eukaryotic cells or to the cytoplasmic membrane of prokaryotic cells. Using genetic engineering techniques, some research groups have therefore tried to secrete intracellular proteins by fusing the gene sequences of consensus signal peptides in-frame to the 5' end of the gene encoding the non-secretion polypeptide. When these heterologous genes were expressed, however, the mere presence of a consensus signal peptide was found to be insufficient for the efficient secretion of non-secretion competent polypeptides across a given biological membrane, a problem which is often encountered in the field of biotechnology. For example, Martens et al. (1995), attached the signal sequence of the juvenile hormone esterase gene to the 5' end of the CryIA(b) insecticidal crystal protein gene to induce secretion but found that secretion into the medium from the insect cells was poor.
A method to efficiently secrete non-secretion competent polypeptides, such as cytoplasmic proteins, nuclear factors and protein subunits would be desirable. This would allow the recombinant protein to be expressed at a higher level. Second because the recombinant protein would be secreted into the extracellular environment, purification of the peptide or protein would not be complicated by the presence of other intracellular proteins and would not involve harming the producing cells.
Advantages of the present invention will become apparent from the following description of the invention with reference to the attached drawings.